Acidic Xylanase(ACX) Activity Assay Kit
Spectrophotometry
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Acidic Xylanase (ACX) Activity Assay Kit - Spectrophotometric Method
Product code: 112889
Package size: 50T
Product Introduction
Xylanase (EC 3.2.1.8)
Xylanase is mainly produced by microorganisms and catalyzes the hydrolysis of xylan. It is also known as pentosanase or hemicellulase. It can break down raw material cell walls and β-glucan, reduce material viscosity in brewing, promote the release of active substances, and reduce non-starch polysaccharides in feed, thereby improving nutrient absorption and utilization.
ACX is generally isolated from acid-tolerant fungi, bacteria, and some molds. It degrades xylan into reducing oligosaccharides and monosaccharides. These products react with 3,5-dinitrosalicylic acid to produce color, with a characteristic absorption peak at 540 nm. The color intensity is proportional to the amount of reducing sugars produced by enzymatic hydrolysis, so ACX activity can be calculated by measuring the increase in absorbance at 540 nm.
Package Contents
| Code | Item | Quantity |
|---|---|---|
| 112889.1 | Reagent I | 1 bottle |
| 112889.2 | Reagent II | 1 bottle |
| 112889.3 | Buffer | 1 bottle |
| 112889.m | Manual | 1 copy |
Quality Standards and Safety Instructions
| Raw Material and Package Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | - | - |
| Reagent II | - | - |
| Buffer | - | - |
Transportation and Storage Conditions
| Item | Condition |
|---|---|
| Transportation | Transport with ice packs. |
| Storage | Store at 2-8 °C, protected from light. |
| Shelf life | 90 days |
Product Instructions
1. Crude Enzyme Extract Preparation
- Fermentation broth: Centrifuge the fermentation broth at 8000 g and 4 °C for 15 min. Collect the supernatant as the sample to be tested.
- Enzyme dry powder: Weigh about 0.1 mg and add 1 mL buffer to dissolve for testing.
- Tissue sample: Prepare according to a tissue mass (g) to extraction solution volume (mL) ratio of 1:5-10. It is recommended to weigh about 0.1 g tissue and add 1 mL buffer. Homogenize in an ice bath, then centrifuge at 8000 g and 4 °C for 10 min. Collect the supernatant for testing.
2. Assay Procedure
Set one control tube for each assay tube.
| Component | Control Tube | Assay Tube |
|---|---|---|
| Sample (µL) | 200 | 200 |
| Buffer (µL) | 300 | 300 |
| Reagent I (µL) | 200 |
Mix well, incubate in a 50 °C water bath for 30 min, then immediately place in a boiling water bath for 10 min to inactivate. Do not let the cap pop open, to prevent water from entering and changing the reaction system.
| Component | Control Tube | Assay Tube |
|---|---|---|
| Reagent I (µL) | 200 | |
| Reagent II (µL) | 300 | 300 |
Mix well and develop color in a boiling water bath for 5 min. Do not let the cap pop open, to prevent water from entering and altering the reaction system. Use a 1 mL glass cuvette to measure absorbance at 540 nm.
Calculate ΔA = Aassay tube- Acontrol tube.
3. Activity Calculation
Standard curve: y = 2.5554x - 0.002, R2= 0.9983
3.1 Calculation Based on Liquid Volume
Enzyme activity definition: Under 50 °C and pH 4.8 conditions, the amount of enzyme required per milliliter of liquid sample per minute to decompose xylan and produce 1 nmol reducing sugar is defined as one unit of acidic xylanase activity.
ACX activity (nmol/min/mL) = (ΔA + 0.002) ÷ 2.5554 ÷ 150 ÷ T × dilution factor × 106= 435 × (ΔA + 0.002)
3.2 Calculation Based on Protein Concentration
Enzyme activity definition: Under 50 °C and pH 4.8 conditions, the amount of enzyme required per milligram of protein per minute to decompose xylan and produce 1 nmol reducing sugar is defined as one unit of acidic xylanase activity.
ACX activity (nmol/min/mg prot) = (ΔA + 0.002) ÷ 2.5554 ÷ 150 ÷ T × dilution factor × 106÷ Cpr = 435 × (ΔA + 0.002) ÷ Cpr
3.3 Calculation on a Fresh Weight Basis
Enzyme activity definition: Under 50 °C and pH 4.8 conditions, the amount of enzyme required per gram of sample per minute to decompose xylan and produce 1 nmol of reducing sugar is one activity unit of acidic xylanase.
ACX activity (nmol/min/g fresh weight) = (ΔA + 0.002) ÷ 2.5554 ÷ 150 ÷ T × dilution factor × 106÷ W = 435 × (ΔA + 0.002) ÷ W
150: molecular weight of xylose
T: reaction time, 30 min
Dilution factor = Vreaction total÷ Vsample= 1000 µL ÷ 200 µL = 5
106: conversion factor, namely 1 mg/mL = 106ng/mL
Cpr: sample protein concentration, mg/mL
W: sample mass, g
Notes
- Before formal determination, select 2-3 samples with large expected differences for pretesting.
- Required instruments and supplies: balance, refrigerated centrifuge, constant-temperature water bath, visible spectrophotometer, 1 mL glass cuvette, and distilled water.
- If Reagent I shows white flocculent or granular precipitates, heat at 60 °C to dissolve before use.
- The absorbance change should be controlled within 0.01-0.8. Otherwise, increase the sample amount or dilute the sample. If dilution is used, adjust the dilution factor in the calculation formula accordingly.
- Store the kit at 2-8 °C. Shelf life is 3 months. It is recommended to use it as soon as possible.
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